Lineage-determining transcription factors constrain cohesin to drive multi-enhancer oncogene regulation.

Multiple enhancers, often located across vast genomic distances, regulate key genes. However, how chromatin topology organization at individual alleles enables cell-type-restricted multi-enhancer gene regulation remains unclear. Using acute protein degradation and time-course population-average chromatin conformation capture in lymphoma, we found that the B-cell- lineage-determining transcription factor EBF1 preferentially positions multiple enhancers at loci containing sparsely distributed genes essential for B-cell identity and oncogenesis. Our time-resolved sub-diffraction optical chromatin architecture tracing of >100,000 alleles in individual lymphoma cells further revealed diverse topological conformations facilitating multi-enhancer interactions. Mechanistically, we found that positioning of enhancers at allelic topological centers is required for their interactions with target promoters, with EBF1 serving as a barrier to the loop-extruding cohesin on enhancers. These findings, which we demonstrate their generalizability to the T-cell-lineage-determining transcription factor TCF1 in T-cell leukemia, suggest that lineage-determining transcription factors radially position enhancers and promoters to enable multi-enhancer regulation of key oncogenes.